Double-pulse laser capture microdissection with a needle beam for reduced collateral damage in biological samples

Laser capture microdissection (LCM) enables spatially selective isolation of cells and tissues, but precise cutting in thick biological samples is constrained by the limited axial interaction range of Gaussian beams and the side-lobe effects of nondiffractive beams. Here, we propose double-pulse laser capture microdissection with a needle beam (DPLM-NB) for thick biological samples. A 60-μm-long needle beam provides an extended axial interaction range, while double-pulse delay control improves energy localization and cutting selectivity. Numerical simulations show that pre-excitation by the first pulse enhances energy coupling in the main-lobe region and weakens the side-lobe-associated response. In 30-μm-thick frozen mouse brain sections, the method reduces the cutting width from 6.75 to 2.47 μm and the threshold energy density from 5.02 to 1.96 J/cm² compared with single-pulse Gaussian-beam cutting. At delays of 1.0–1.3 ns, it achieves an average cutting width of 2.63 μm and threshold energy density of 1.85 J/cm² with reduced collateral damage, enabling precise microdissection for downstream omics analysis.